Optimization of N'-(arylsulfonyl)pyrazoline-1-carboxamidines by exploiting a novel interaction site in the 5-HT6 antagonistic binding pocket.

The discovery of non-basic N'-(arylsulfonyl)pyrazoline-1-carboxamidines as 5-HT6 antagonists with unique structural features was recently disclosed. Here we describe how this structural class was further developed by addressing an unexplored interaction site of the 5-HT6 receptor. Compound 13 resulting from this effort is a highly potent and selective 5-HT6 antagonist with improved metabolic stability. It is furthermore devoid of hERG affinity. Despite its modest CNS/plasma ratio, a high brain free fraction ensured substantial exposure to allow for rodent cognition studies.

Identification and development of biphenyl substituted iminosugars as improved dual glucosylceramide synthase/neutral glucosylceramidase inhibitors.

This work details the evaluation of a number of N-alkylated deoxynojirimycin derivatives on their merits as dual glucosylceramide synthase/neutral glucosylceramidase inhibitors. Building on our previous work, we synthesized a series of D-gluco and L-ido-configured iminosugars N-modified with a variety of hydrophobic functional groups. We found that iminosugars featuring N-pentyloxymethylaryl substituents are considerably more potent inhibitors of glucosylceramide synthase than their aliphatic counterparts. In a next optimization round, we explored a series of biphenyl-substituted iminosugars of both configurations (D-gluco and L-ido) with the aim to introduce structural features known to confer metabolic stability to drug-like molecules. From these series, two sets of molecules emerge as lead series for further profiling. Biphenyl-substituted L-ido-configured deoxynojirimycin derivatives are selective for glucosylceramidase and the nonlysosomal glucosylceramidase, and we consider these as leads for the treatment of neuropathological lysosomal storage disorders. Their D-gluco-counterparts are also potent inhibitors of intestinal glycosidases, and because of this characteristic, we regard these as the prime candidates for type 2 diabetes therapeutics.

N'-(arylsulfonyl)pyrazoline-1-carboxamidines as novel, neutral 5-hydroxytryptamine 6 receptor (5-HT6R) antagonists with unique structural features.

The 5-HT(6) receptor (5-HT(6)R) has been in the spotlight for several years regarding CNS-related diseases. We set out to discover novel, neutral 5-HT(6)R antagonists to improve off-target selectivity compared to basic amine-containing scaffolds dominating the field. High-throughput screening identified the N'-(sulfonyl)pyrazoline-1-carboxamidine scaffold as a promising neutral core for starting hit-to-lead. Medicinal chemistry, molecular modeling, small molecule NMR and X-ray crystallography were subsequently applied to optimize the leads into antagonists (compounds 1-49) displaying high 5-HT(6)R affinity with optimal off-target selectivity. Unique structural features include a pseudoaromatic system and an internal hydrogen bond freezing the bioactive conformation. While physicochemical properties and CNS availability were generally favorable, significant efforts had to be made to improve metabolic stability. The optimized structure 42 is an extremely selective, hERG-free, high-affinity 5-HT(6)R antagonist showing good human in vitro metabolic stability. Rat pharmacokinetic data were sufficiently good to enable further in vivo profiling.

Synthesis of spirohydantoins and spiro-2,5-diketopiperazines via resin-bound cyclic alpha,alpha-disubstituted alpha-amino esters.

A seven-step solid-phase synthesis of spirohydantoins and an eight-step solid-phase synthesis of spiro-2,5-diketopiperazines is reported. Key intermediate in the synthesis of both compound libraries is the resin-bound cyclic alpha,alpha-disubstituted alpha-amino ester, which can be obtained after selective homogeneous reduction of the aliphatic nitro ester using tin(II) chloride dihydrate. Nitro ester, in turn, is synthesized by a high-pressure-assisted [4 + 2] cycloaddition of resin-bound nitro alkene and butadiene, whereas nitro alkene is obtained by a Knoevenagel condensation of resin-bound nitro acetate with an imine. Novel spirohydantoins are obtained by isocyanate coupling with the resin-bound amino ester 5, followed by cyclization cleavage using a base. Novel spiro-2,5-diketopiperazines are obtained by PyBOP coupling of a Fmoc-protected amino acid with resin-bound amino ester, followed by Fmoc deprotection and an acid-assisted cyclization cleavage. After preparation of seven different resin-bound alpha,alpha-disubstituted alpha-amino esters, a 7 x 8 compound library of spirohydantoins was synthesized using eight different isocyanates, and a 7 x 8 compound library of spiro-2,5-diketopiperazines was synthesized using eight different Fmoc amino acids.

Function of the ABC transporters, P-glycoprotein, multidrug resistance protein and breast cancer resistance protein, in minimal residual disease in acute myeloid leukemia.

BACKGROUND AND OBJECTIVES: Relapse is common in acute myeloid leukemia (AML) because of persistence of minimal residual disease (MRD). ABC-transporters P-glycoprotein (Pgp) and multidrug resistance protein (MRP), are thought to contribute to treatment failure, while it is unknown whether breast cancer resistance protein (BCRP) does so. However, whether up-regulation of pump activity or selection of subpopulations with higher pump activity occurs during chemotherapy is unclear. The aim of this study was to elucidate whether ABC-transporter function changes during the course of disease. DESIGN AND METHODS: MRD cells were identified using leukemia-associated phenotypes combined with a fluorescent probe assay with substrate/modulator: Syto16/ PSC833 (Pgp), calcein-AM/probenecid (MRP) and BODIPY-prazosin/Ko143 (BCRP); efflux profiles were directly compared with blasts at diagnosis and relapse from the same patient. RESULTS: At diagnosis BCRP activity was undetectable in AML blasts from 23/26 cases, while Pgp activity was present in 36/45 and MRP activity in 26/44 of the cases. Furthermore, no subpopulations of blasts with considerably higher drug efflux capacities were found. Overall, no consistent changes were observed at follow-up [during chemotherapy (n=20), MRD (n=37), relapse (n=26))] in forty-five patients, the mean activities (as percentages of values at diagnosis) were 97% (Pgp), 103% (MRP) and 102% (BCRP). INTERPRETATION AND CONCLUSIONS: Emergence of MRD is thus not accompanied by either upregulation of ABC-transporter function during or after chemotherapy or by selection of pre-existing highly resistant subpopulations. The prognostic value of Pgp and MRP is, therefore, likely related to drug efflux capacity homogeneously distributed in the whole blast population, while BCRP probably has a limited function in drug efflux-related resistance in AML.

Potent and specific inhibition of the breast cancer resistance protein multidrug transporter in vitro and in mouse intestine by a novel analogue of fumitremorgin C.

Inhibitors of the breast cancer resistance protein (BCRP/ABCG2) multidrug transporter are of interest as chemosensitizers for clinical drug resistance, for improving the pharmacokinetics of substrate chemotherapeutic drugs, and in functional assays of BCRP activity for tailoring chemotherapy. The fungal toxin fumitremorgin C (FTC) is a potent and specific inhibitor of BCRP, but its neurotoxic effects preclude use in vivo. We have therefore evaluated a new tetracyclic analogue of FTC, Ko143, as a practical inhibitor of BCRP, comparing it with two other analogues in the same class and with GF120918. All three FTC analogues are effective inhibitors of both mouse Bcrp1 and human BCRP, proving highly active for increasing the intracellular drug accumulation and reversing Bcrp1/BCRP-mediated multidrug resistance. Indeed, Ko143 appears to be the most potent BCRP inhibitor known thus far. In contrast, the compounds have only low activity against P-glycoprotein, the multidrug resistance-associated protein (MRP1), or other known drug transporters. They are nontoxic in vitro at useful concentrations and evinced no signs of toxicity in mice at high oral or i.p. doses. Administered p.o. to inhibit intestinal Bcrp1, Ko143 markedly increased the oral availability of topotecan in mice. It is thus the first highly potent and specific BCRP inhibitor applicable in vivo. As such, Ko143 and other FTC analogues of this type represent valuable reagents for analysis of drug resistance mechanisms and may be candidates for development as clinical BCRP inhibitors.